Establishment of A Mouse Sepsis-Induced Immunosuppression Model By Intranasal Instillation of Bacteria

Summary

Here, we present a protocol for establishing a mouse sepsis immunosuppression model via intranasal instillation of Klebsiella pneumoniae and validated the model through various indicators.

Abstract

Sepsis is a life-threatening organ failure caused by the host's inappropriate response to infection. Pneumonia is the primary cause of sepsis. Animal models are important tools for studying the pathogenesis of sepsis and evaluating new treatment strategies. Previous animal models of sepsis lacked natural infection pathways, making it difficult to accurately reflect the clinical infection process.

This study introduced a mouse sepsis immunosuppression model established by intranasal instillation of Klebsiella pneumoniae (KP) to simulate the natural respiratory infection pathway. After modeling with different concentrations of KP, the inflammation of the model was evaluated at 12 h for cytokine interleukin-6 (IL-6) levels, and organ damage was evaluated at 24 h for alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), and blood urea nitrogen (BUN) levels. The survival rate of the model was analyzed after 7 days. The percentage of lymphocytes and white blood cells, the proportion of major histocompatibility complex class II (MHC-II) expression in monocytes, and the mean fluorescence intensity (MFI) of IL-10 and TGF-β1 in CD4⁺ T cells were observed to evaluate the immune suppression in mice. After 7 days of modeling, a secondary infection was performed by intranasal instillation of Pseudomonas aeruginosa (PA). The bacterial load in bronchoalveolar lavage fluid (BALF) 24 h after secondary infection and the mortality rate within 14 days were assessed to further evaluate the model's immune suppression.

Nasal instillation of high-concentration Klebsiella pneumoniae can effectively induce an immune suppression. Its purpose is to simulate sepsis induced by a natural respiratory infection pathway and to provide a standard model for sepsis research in animals.

Introduction

Sepsis is a life-threatening organ failure resulting from the host's inappropriate response to infection^1,2,3. It is one of the leading causes of death in intensive care units (ICUs). Globally, there are 48.9 million sepsis cases annually, with 11 million associated deaths⁴. Despite recent advancements in anti-infective therapy and organ support technologies, the pathogenesis of sepsis remains incompletely understood, and specific treatment options are lacking. As such, sepsis has become a key focus and challenge in clinical medical research

Protocol

All experimental procedures described in this article were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University (SCXK(PLA)20120031).

1. Experimental animals

1. Use C57BL/6 mice, male, aged 8–10 weeks, weighing 23–28 g (Animal License No.: SCXK (Xiang) 2019-0004). House the mice in a specific pathogen-free (SPF) environment with free access to food and water. Maintain the housing conditions as follows: room temperature of (24 ± 2ºC), humidity of 50%–55%, and a 12 h/12 h light-dark cycle.

2. Bacteri....

Results

At 12 h after intranasal instillation, the serum IL-6 level in the 1 × 10⁸ KP group was significantly increased (p < 0.0001, n = 5) (Figure 2A). At 24 h after intranasal instillation, the serum levels of ALT (p < 0.01, n = 5) (Figure 2B), AST (p < 0.01, n = 5) (Figure 2C), CK (p < 0.01, n = 5) (Figure 2D), and BUN (p < 0.001, n = 5) (Figure 2E) in .......

Discussion

Sepsis is a complex clinical syndrome, and the characteristic immunosuppressive state during its late stage is a key factor leading to patient death^12,13. Therefore, preclinical models that accurately simulate the pathophysiology of sepsis-induced immunosuppression are crucial for in-depth exploration of its mechanisms and the development of new therapies. In this study, we successfully established and validated a mouse sepsis model induced by intranasal instilla.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biochemical incubator	YSEI	SHH-150L	Bacterial incubation
C57BL/6J mice	Vital River Laboratories	8-10 weeks, male
CD116b	BioLegend	101257	Flow Antibodies (Anti-Mouse)
CD25	BD bioscience	740447	Flow Antibodies (Anti-Mouse)
CD3	BioLegend	100203	Flow Antibodies (Anti-Mouse)
CD4	BioLegend	100544	Flow Antibodies (Anti-Mouse)
CD45	BioLegend	103116	Flow Antibodies (Anti-Mouse)
CD8	BioLegend	100752	Flow Antibodies (Anti-Mouse)
Cell stain buffer	BioLegend	420201
Constant temperature shaker	Honour	HNY-100B	Bacterial cultivation
Flow cytometer	Beckman Coulter	CytoFLEX	Flow cytometry analysis
High-speed centrifuge	Beckman Coulter	AIIEGRA
IDEXX catalyst one	IDEXX	Catalyst One	Biochemical analysis
IL-10	BioLegend	505026	Flow Antibodies (Anti-Mouse)
Insulin cyringe	WEGO	1mL	Blood collection
Klebsiella pneumoniae ATCC 43816	biobw.org	bio-81659
LB Broth (powder)	Solarbio	L1010	LB liquid culture medium
LB Broth (powder)	Solarbio	L1015	LB agar plate
LY6C	BioLegend	128010	Flow Antibodies (Anti-Mouse)
Ly6G	BioLegend	127606	Flow Antibodies (Anti-Mouse)
MHC-II	BioLegend	107622	Flow Antibodies (Anti-Mouse)
Micropipette	Eppendorf	P100
Red blood cell lysis buffer	Solarbio	R1010
Spectrophotometer	Eppendorf	Eppendorf6131	Optical density measurement
Statistical software	GraphPad Software	Graphpad Prism10.0
Sterile and enzyme-free pipette tips	Biosharp	200μL
TF diluent buffer	BD Pharmingen	51-90008101
TF fix/perm buffer	BD Pharmingen	51-90008100
TF perm/wash buffer	BD Pharmingen	51-90008102
TGF-β1	BioLegend	141406	Flow Antibodies (Anti-Mouse)
Varioskan LUX multimodemicroplate reader	Thermo Scientific	Varioskan LUX